Dopamine is important in Parkinson's disease and the action of abused drugs (e.g. amphetamines) and clinical psychiatric drugs (e.g. antipsychotics). Because dopamine cannot be seen directly in living neurons with the light microscope, it is difficult to localize and quantify Ca2+-dependent vesicular and amphetamine- induced nonvesicular dopamine release from the soma, dendrites and terminals in living brain tissue. Likewise, the hypothesis that drugs accumulate in and are released from monoamine vesicles has not been tested. Such corelease of antipsychotic drugs with their target transmitters would result in concentrated drug delivery when and where drug action is needed (i.e., at active dopamine and serotonin synapses) resulting in greater efficacy and specificity. To be able to visualize dopamine and drug dynamics in living neurons, we have been developing new experimental approaches based on multiphoton microscopy in the rodent brain slice. First, we found that the clinically used anxiolytic antipsychotic drug cyamemazine produces visible fluorescence upon multiphoton excitation. Multiphoton imaging in midbrain slices showed that cyamemazine is subject to acidic trapping and Ca2+-dependent release. Second, multiphoton microscopy detected autofluorescence in substantia nigra dopamine neurons. Amphetamine induced dopamine transporter- mediated depletion of this signal. Likewise, depolarization induced Ca2+-dependent depletion. These results support the hypothesis that intrinsic multiphoton autofluorescence is derived from dopamine. This proposal builds on these preliminary results to first determine whether multiphoton microscopy in the brain slice can image a dopamine-derived signal that reveals content and release. Then the hypothesis that there is colocalized release of an antipsychotic drug with its target monoamine transmitters (i.e., dopamine and serotonin) is tested. These experiments will explore new optical approaches for studying dopamine and drugs in living brain tissue. Furthermore, determining whether there is corelease of an anxiolytic antipsychotic drug with serotonin and dopamine would be important for establishing a new paradigm for psychiatric drug action.